HIV-1 gp41 contains highly conserved sequence elements that are targets for viral entry inhibitors and broadly neutralizing, monoclonal antibodies (mAbs). The broadly HIV-l-neutralizing mAbs, 2F5, 4E10 and Z13, bind to the membrane-proximal external region (MPER) of gp41. Although the MPER, and gp41 more generally, are targets for HIV-1 vaccine development, immunizations of animals using gp41 or antigens bearing neutralizing epitopes of gp41 typically have not elicited significant titers of neutralizing Abs against primary HIV-I. Moreover, the N- and C-heptad repeat (HR) regions of gp41 appear to be accessible to various HIV-1 entry inhibitors, but due to their transient exposure during HIV-1 entry into cells, the degree of accessibility by Ab to these otherwise susceptible sites remains an open question in HIV-1 vaccine development. This proposal is to generate panels of mAbs against the epitopes of the neutralizing mAbs 2F5, 4E10 and Z13, and against sites of gp41 to which HIV-1 entry inhibitors bind including the N- and C-HR regions. MAbs will be generated using phage display technology in three different ways. In the first approach, single chain Fvs will be generated from rabbits immunized with peptides from the MPER and the N- and C- HR regions of gp41. In the second approach, in vitro evolution techniques will be used to select variants of Fabs Z13 and 2F5 with antigen-binding profiles that differ from the wild-type Fab, and the variants will be used as tools to better understand the neutralizing activity of these mAbs. Finally, a panel of sera from HIV-1 infected individuals will be screened with gp41 peptides from the MPER and the N- and C- HR regions of gp41, and a Fab phage library will be prepared from the immune repertoire of a patient with favorable serum characteristics. Fine epitope mapping and HIV-1 neutralization assays will be used to characterize the anti-gp41 mAbs that are selected in all three approaches. A panel of mAbs against the described epitopes on gp41 will provide valuable tools to aid in HIV-1 vaccine design.